A variety of expression vectors have heretofore been developed for using in the production of recombinant proteins. In particular, for the expression systems utilizing microorganisms such as Escherichia coli and yeast as hosts, there have been provided those which are expected to give high yields. In the case of proteins whose biological activity depends on sugar chains, it is necessary to produce such proteins by using animal cells as the host. In this regard, recently, a vector which permits a high level expression has also been developed (JP 10-179169 A), and there is an example of successful expression of human mannan binding protein by using this vector.
Thus, systems utilizing Escherichia coli, yeast or animal cells have been used by many investigators in order to produce foreign proteins. In the systems utilizing Escherichia coli as the host, expressing capacity can be enhanced by using a potent promoter derived from Escherichia coli. However, in most cases, foreign proteins expressed accumulate within cells as inclusion bodies. Therefore, it is necessary to solubilize the protein by using a denaturing agent such as urea and guanidine and then to unwind the protein to the native form. Then, it is extremely difficult to directly isolate and purify the protein in the active form, and complicated procedures are required.
Further, in the system utilizing yeast as the host, a proteolytic degradation is unavoidable. Then, improvement in the expression of soluble proteins can not be expected. In addition, the proteins are modified in a different way because of remarkably different expressing environment from the intercellular environment of higher animals. Furthermore, although systems utilizing animal cells may allow the production of recombinant proteins in forms comparable to natural ones, complicated procedures are needed, thereby having a drawback with respect to production efficiency.
In recent years, an expression system has received an attention, wherein insect cells are used as the host infected with a baculovirus. The reason for this is, for example, that the baculovirus, upon infecting insect cells, produces more than approximately 25% of the total cell protein as a polyhedron protein, and a high expression system for foreign proteins has been developed by using this potent promoter. And, the following advantages have been recognized in regard to the production of foreign proteins by using a baculovirus-insect cell expression system: (a) the expression levels of foreign proteins are high; (b) processing of signal peptides, modification with sugar chains, phosphate, lipids, etc., dimerization, virion formation, intron splicing, and the like take place as those in natural proteins; (c) the intracellular localization of protein within insect cells is the same as that with the natural protein; (d) insect cells can be cultivated in a suspension culture.
Heretofore, a variety of proteins (e.g., insulin, interferons, erythropoietin, mannan binding protein, conglutinin, etc.) have been produced in insect cells and animal cells by using gene engineering technology. In order to obtain recombinant proteins with quality comparable to that of the natural form, an expression system utilizing animal cells (e.g., mammalian cells or insect cells) as hosts is essential as described above. Then, the development of expression vectors which are useful in said expression system has been desired.
The development of expression vectors has been attempted primarily along two approaches, namely an attempt to enhance the expression level of recombinant proteins, and an attempt to simplify the purification of expressed recombinant proteins. Vectors which aim at enhancing the expression level include, for example, the vector disclosed in JP 10-179169 A. As vectors which aim at enhancing the purification efficiency, histidine Tag vector (manufactured by Invitrogen Corporation) is known.
pSecTag vector (manufactured by Invitrogen Corporation) is commercially available as a vector which facilitates purification of recombinant proteins secreted extracellularly. This vector is used with animal cells as the host, and contains a secretory signal, a multicloning site capable of inserting a nucleotide sequence encoding a target protein, a myc epitope which recognizes a fusion protein, and a polyhistidine Tag which allows purification of the protein by a nickel chelate resin. However, this vector can not express a target protein in insect cells. Also, even if a protein is expressed in animal cells, amino acids such as myc epitope and histidine Tag are added to the C-terminus of a target protein, precluding the protein from being obtained as a pure recombinant protein, which is a drawback of using this vector.
On the other hand, pFastBAC HT vector (manufactured by GIBCO BRL) is commercially available as a vector which enables proteins to be expressed in insect cells and to be purified easily. This vector uses insect cells as the host and contains a histidine Tag nucleotide sequence, a cleavable nucleotide sequence which allows the cleavage of the sequence between that encoding the histidine Tag sequence and that encoding a target protein, and a multicloning site capable of inserting the nucleotide sequence encoding the target protein. However, this vector does not contain a secretory signal which enables extracellular secretion of a target protein to. Therefore, cells must be disrupted in order to obtain a target protein expressed intracellularly. A myriad of proteins within the cells will be released by cell disruption, making it extremely difficult to purify the target protein.
Also, it is desirable that an expressible recombinant protein is identical to the corresponding natural protein in its amino acid sequence, with no expression vector-derived amino acids being added to the C-terminus or the N-terminus. In particular, it has been known that the type of the amino acid at position 1 (N-terminus) of a natural or recombinant protein markedly affects the stability of said protein. That is, there is a strong correlation between the property of the N-terminal amino acid and the in vivo half life of the protein, which is designated as the N-end rule. This correlation holds true to a greater or lesser extent with proteins of every living system that has been so far studied spanning from bacteria to mammals.
Under the above-described circumstances, it has been desired to develop an expression vector that can express recombinant proteins in an expression system which can utilize animal cells, mammalian cells or insect cells in particular, as the host and can secrete the protein extracellularly, wherein the obtained recombinant can be purified by a simple procedure, and still further at least the N-terminus of the amino acid sequence of the recombinant protein is identical to that of the natural protein.